US 7109347 B2
The present invention provides new uses of DPIV-inhibitors of the present invention, and their corresponding pharmaceutically acceptable acid addition salt forms, for treating conditions mediated by DPIV or DPIV-like enzymes, such as cancer and tumors. In a more preferred embodiment, the compounds of the present invention are useful for the treatment of metastasis and tumor colonization.
1. A method for treating breast cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of at least one inhibitor of dipeptidyl peptidase IV (DPIV), wherein said inhibitor is an amino acid linked to a thiazolidine or a pyrrolidine group by a peptide bond.
2. The method according to
3. The method according to
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5. A method according to
This application is a continuation of Ser. No. 10/172,809 filed Jun. 13, 2002, now abandoned.
This application claims the priority of U.S. provisional application U.S. 60/301,158 entitled Peptide Structures Useful for Competitive modulation of Dipeptidyl Peptidase IV Catalysis filed on Jun. 27, 2001. Priority is also claimed from U.S. provisional application U.S. 60/360,909 entitled Glutaminyl-based DPIV Inhibitors filed on Feb. 28, 2002. This application also claims the priority of the following foreign applications EP 01 114 796.4 entitled Peptide Structures Useful for Competitive Modulation of Dipeptidyl Peptidase IV Catalysis having a priority date of Jun. 27, 2001, DE 101 50 203.6 entitled Peptidylketone als Inhibitoren der DPIV having a priority date of Oct. 12, 2001 and DE 101 54 689.0 entitled Substituierte Aminoketonverbindungen having a priority Date of Nov. 9, 2001. The above applications are incorporated in their entirety by reference.
The present invention relates to inhibitors of dipeptidyl peptidase IV and dipeptidyl peptidase IV-like enzyme activity and, more particularly, pharmaceutical compositions containing said compounds, and the use of said compounds for the treatment of cancer and tumors. The present invention especially provides a method for the inhibition of metastasis and tumor colonization.
Dipeptidyl peptidase IV (DPIV) is a serine protease which cleaves N-terminal dipeptides from a peptide chain containing, preferably, a proline residue in the penultimate position. Although the biological role of DPIV in mammalian systems has not been completely established, it is believed to play an important role in neuropeptide metabolism, T-cell activation, attachment of cancer cells to the endothelium and the entry of HIV into lymphoid cells.
Likewise, it has been discovered that DPIV is responsible for inactivating glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide also known as gastric-inhibitory peptide (GIP). Since GLP-1 is a major stimulator of pancreatic insulin secretion and has direct beneficial effects on glucose disposal, in WO 97/40832 and U.S. Pat. No. 6,303,661 inhibition of DPIV and DPIV-like enzyme activity was shown to represent an attractive approach for treating non-insulin-dependent diabetes mellitus (NIDDM).
The present invention provides a new use of DPIV-inhibitors for the treatment of conditions mediated by inhibition of DPIV and DPIV-like enzymes, in particular the treatment of cancer and tumors and the inhibition of metastasis and tumor colonization, and pharmaceutical compositions e.g. useful in inhibiting DPIV and DPIV-like enzymes and a method of inhibiting said enzyme activity.
This invention relates to a method of treatment, in particular to a method for the treatment of cancer, tumors, metastasis and tumor colonization and to compositions for use in such method. Dipeptidyl peptidase IV (DPIV) is a post-proline (to a lesser extent post-alanine, post-serine or post-glycine) cleaving serine protease found in various tissues of the body including kidney, liver, and intestine.
It is known that DPIV-Inhibitors may be useful for the treatment of impaired glucose tolerance and diabetes mellitus (International Patent Application, Publication Number WO 99/61431, Pederson R A et al, Diabetes. 1998 August; 47(8):1253–8 and Pauly R P et al, Metabolism 1999 March; 48(3):385–9). In particular WO 99/61431 discloses DPIV-Inhibitors comprising an amino acid residue and a thiazolidine or pyrrolidine group, and salts thereof, especially L-threo-isoleucyl thiazolidine, L-allo-isoleucyl thiazolidine, L-threo-isoleucyl pyrrolidine, L-allo-isoleucyl thiazolidine, L-allo-isoleucyl pyrrolidine, and salts thereof.
Further examples of low molecular weight dipeptidyl peptidase IV inhibitors are agents such as tetrahydroisoquinolin-3-carboxamide derivatives, N-substituted 2-cyanopyroles and -pyrrolidines, N-(N′-substituted glycyl)-2-cyanopyrrolidines, N-(substituted glycyl)-thiazolidines, N-(substituted glycyl)-4-cyanothiazolidines, amino-acyl-borono-prolyl-inhibitors, cyclopropyl-fused pyrrolidines and heterocyclic compounds. Inhibitors of dipeptidyl peptidase IV are described in U.S. Pat. No. 6,380,398, U.S. Pat. No. 6,011,155; U.S. Pat. No. 6,107,317; U.S. Pat. No. 6,110,949; U.S. Pat. No. 6,124,305; U.S. Pat. No. 6,172,081; WO 95/15309, WO 99/61431, WO 99/67278, WO 99/67279, DE 198 34 591, WO 97/40832, DE 196 16 486 C 2, WO 98/19998, WO 00/07617, WO 99/38501, WO 99/46272, WO 99/38501, WO 01/68603, WO 01/40180, WO 01/81337, WO 01/81304, WO 01/55105, WO 02/02560 and WO 02/14271, the teachings of which are herein incorporated by reference in their entirety.
The term DPIV-like enzymes relates to structurally and/or functionally DPIV/CD26-related enzyme proteins (Sedo & Malik, Dipeptidyl peptidase IV-like molecules: homologous proteins or homologous activities? Biochimica et Biophysica Acta 2001, 36506: 1–10). In essence, this small group of enzymes has evolved during evolution to release H-Xaa-Pro-Dipeptides and H-Xaa-Ala-Dipeptides from N-terminus of oligo- or polypeptides. They show the common feature, that they accomotate in the Pro-position also Al, Ser, Thr and other amino acids with small hydrophobic side-chains as, Gly or Val. The hydrolytic efficacy is ranked Pro>Ala>>Ser, Thr>>Gly, Val. Same proteins have been only available in such small quantities, that only the post-Pro or post-Ala cleavage could be established. While the proteins: DPIV, DP II, FAPα (Seprase), DP 6, DP 8 and DP 9 are structurally related and show a high sequence homology, attractin is an extraordinary functional DPIV-like enzyme, characterized by a similar activity and inhibitory pattern.
Further DPIV-like enzymes are disclosed in WO 01/19866, WO 02/04610, WO 02/34900 and WO 02/31134. WO 01/19866 discloses novel human dipeptidyl aminopeptidase (DPP8) with structural und functional similarities to DPIV and fibroblast activation protein (FAP). WO 02/04610 provides reagents, which regulate human dipeptidyl peptidase IV-like enzyme and reagents which bind to human dipeptidyl peptidase IV-like enzyme gene product. These reagents can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, tumors and peripheral and central nervous system disorders including pain and neurodegenerative disorders. The dipeptidyl peptidase IV-like enzyme of WO 02/04610 is well known in the art. In the Gene Bank data base, this enzyme is registered as KIAA1492 (registration in February 2001, submitted on Apr. 4, 2000, AB040925). In the Merops data base, the dipeptidyl peptidase IV-like enzyme of WO 02/04610 is registered as non-protease homologue. The Merops homologue of the dipeptidyl peptidase IV-like enzyme disclosed in WO 02/04610 and the active site motive thereof was confirmed by the human genome project. WO 02/34900 discloses a novel dipeptidyl peptidase 9 (DPP9) with significant homology with the amino acid sequences of DPIV and DPP8. WO 02/31134 discloses three DPIV0-like enzymes. DPRP1, DPRP2 and DPRP3. Sequence analysis revealed, that DPRP1 is identical to DPP8, as disclosed in WO 01/19868, that DPRP2 is identical to DPP9 and that DPRP3 is identical to KIAA1492 as disclosed in WO 02/04610.
DPIV and DPIV-Like Enzymes in Immunophysiology and Cancer
Dipeptidyl peptidase IV (DPIV; EC 126.96.36.199; CD26) CD26 is a M r 110,000 surface glycoprotein with an array of diverse functional properties that is expressed on a number of tissues, including epithelial cells and leukocyte subsets (Mentlein, 1999). Furthermore, it is a membrane-associated ectopeptidase that possesses DPIV-like activity in its extracellular domain and is able to cleave N-terminal dipeptides from polypeptides with either L-proline or L-alanine in the penultimate position. In general, DPIV is recognized as an ectopeptidase with a triple functional role. DPIV is involved in catalyzing the release of Xaa-Pro dipeptides from circulating hormones and chemokines (De Meester et al, 1999; Mentlein, 1999), in T cell dependent immune responses (Kähne et al, 1999; Korom et al, 1997), and in cell adhesion including metastasis (Mentlein, 1999).
In addition DPIV has been identified as the ADA binding protein, thereby regulating ADA surface expression, with the DPIV/ADA complex perhaps playing a key role in the catalytic removal of local adenosine to regulate immune system function. Besides being a key immunoregulatory molecule, DPIV may have a potential role in the development of certain neoplasms (Mattern et al., 1993; Carbone et al., 1995). In eukaryotic cells, cell cycle progression is controlled at the G1-S checkpoint by a group of related enzymes known as the CDKs, which are positively regulated by their physical association with regulatory subunits called cyclins. It has been demonstrated that binding of soluble anti-CD26 antibodies inhibits the growth of anaplastic large cell T-cell lymphoma cell lines, both in in vitro and in vivo experiments (Ho et al., 2001).
Cancer is a group of over 150 diseases characterized by the uncontrolled growth of abnormal cells in the body. Normal cells can become abnormal when they are exposed to carcinogens such as radiation or particular drugs or chemicals. They can also turn malignant (cancerous) when they are attacked by certain viruses or when some not-yet-fully-understood internal signal occurs. Once cells become malignant, they multiply more rapidly than usual. Then they often form masses called tumors that invade nearby tissue and interfere with normal bodily functions. Cancer cells also have a tendency to spread to other parts of the body, where they may form a secondary tumor.
Mechanisms of Metastasis
The outcome of cancer metastasis depends on multiple interactions within the target tissue and depends on the microenvironment including cellular adhesion molecules (Carlos, 2001), chemokines (Muller et al., 2001), or hydrodynamic effects (Haier and Nicholson, 2001) and many other factors (Fidler, 2001). In addition, a very rapid attraction of leukocytes and specific cellular responses at the tumor sites may play a critical role in the early host defense against cancer (Shingu et al.; 2002). These early changes may be of critical importance for the outcome of metastatic disease and may extend the present understanding of the host resistance against metastasis.
DPIV and DPIV-Like Enzymes and Tumor Adhesion and Colonization
For cancer cell or metastatic cell adhesion, a prominent expression of DPIV on endothelia of lung capillaries accounts for arrest of blood borne breast cancer cells (Johnson et al, 1993). Fibronectin (FN) and probably also collagen collected on the breast cancer cell surface were identified as the principal ligands for DPIV (Abdel-Ghany et al, 1998; Cheng et al, 1998).
Ho and colleagues (2001) show that binding of soluble anti-CD26 monoclonal Ab 1F7 inhibits the growth of the human CD301 anaplastic large cell T-cell lymphoma cell line Karpas 299 in both in in vitro and in vivo experiments. In vitro experiments show that 1F7 induces cell cycle arrest at the G1-S checkpoint, associated with enhanced p21 expression that is dependent on de novo protein synthesis. Furthermore, experiments with a severe combined immunodeficient mouse tumor model demonstrate that 1F7 treatment significantly enhances survival of tumor-bearing mice by inhibiting tumor formation.
Protease Inhibitors, Antibodies and Proteases as Anti-Tumor Agents
WO 95/29691 discloses proline phosphonate derivatives as inhibitors of serine proteases with chymotrypsin-like, trypsin-like, elastase-like and dipeptidyl peptidase IV specificity and their roles as anti-inflammatory agents, anticoagulants, anti-tumor agents and anti-AIDS agents.
WO 98/53812 and WO 97/48409 disclose novel methods of using phosphonate derivatives, hydroxyphosphinyl derivatives, and phosphoramidate derivatives to inhibit N-Acetlyated α-Linked Acidic Dipeptidase (NAALADase) enzyme activity, and to treat prostate diseases, especially using these compounds for the inhibition of prostate cancer cell growth.
WO 01/92273 discloses new benzenedicarboxylic acid derivative compounds, pharmaceutical compositions, diagnostic methods and diagnostic kits that include those compounds and methods of using those compounds for inhibiting NAALADase enzyme activity, detecting diseases where NAALADase levels are altered, affecting neuronal activity, affecting TGF-β activity, inhibiting angiogenesis and treating glutamate abnormalities, neuropathy, pain, compulsive disorders, prostate diseases, cancer and glaucoma.
WO 01/34596 discloses pyrrolecarbonylimino derivatives, pharmaceutical compounds and methods of using those compounds to inhibit NAALADase enzyme activity, thereby affecting neuronal activities, inhibiting angiogenesis and treating glutamate abnormalities, compulsive disorders, prostate diseases and cancer.
WO 00/71135 discloses a method for treating subjects with abnormal cell proliferation. The method involves administering to subjects in need of such treatment an effective amount of boro-proline compounds, to inhibit cell proliferation such as that associated with tumor growth and metastasis. A method for inhibiting angiogenesis in an abnormal proliferative cell mass by the administration of a boro-proline derivative is also provided. The invention of WO 00/71135 is based, in part, on the observation, that the boro-proline derivatives are able to inhibit the enzymatic activity of fibroblast activation protein-alpha (FAP-α).
WO 00/71571 relates to a prodrug that is capable of being converted into a drug by the catalytic action of human fibroblast activation protein-alpha (FAP-α), said prodrug having a cleavage site which is recognised by FAP-α, and said drug being cytotoxic or cytostatic under physiological conditions. These prodrugs are converted into a drug at the site of the tumor.
WO 00/10549 discloses compounds and a method for regulation of substrate activity in vivo useful for the treatment of medical disorders such as arteriosclerosis, allergies, inflammation, angiogenesis, cardiogenesis, neoplasm, tumor, cancer, a hepatic disease, an intestinal disease, organ vascularization, and microbial and viral infections. The compounds consist of a targeting moiety that binds to DPIV, and a reactive group, that reacts at a reactive center of DPIV. Said compounds are used to prevent chemokine alteration by inhibiting DPIV activity.
WO 00/36420 discloses a method for identifying nucleotide sequences that are differentially expressed in tumor cells, preferably primary breast tumor cells, comprising exposing a tumor cell containing tissue sample to an agent specific for fibroblast activation protein (FAP) separating cells recognised by said agent from the remaining cells in the sample and harvesting said remaining cells. Nucleic acid molecules derived from the use of this technique are also described, together with compositions comprising the same and their uses in pharmaceutical compositions for treating a disease, preferably breast cancer.
WO 99/47152 discloses a method of suppressing the malignant phenotype or inducing apoptosis of cancer cells in a subject, comprising introducing into the cancer cell an amount of a nucleic acid encoding a dipeptidyl peptidase IV protein or fibroblast activating protein-α, thereby suppressing the malignant phenotype of the cancer. WO 99/47152 also discloses a method of inducing expression of dipeptidyl peptidase IV or fibroblast activating protein-α in cancer cells of a subject, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an agent capable of activating transcription of the dipeptidyl peptidase IV gene or fibroblast activating protein-α gene and a pharmaceutical acceptable carrier or diluent.
WO 01/74299 discloses antibodies that specifically bind to a membrane protease complex, the complex consisting of two homodimers of seprase and dipeptidyl peptidase IV (DPIV), obtained from mammalian, preferably human cell membranes. The antibodies specifically bind to the DPIV protease of the seprase-DPIV complex. This membrane protease complex resides on cell surface invadopodia at the leading edge of angiogenic endothelia, migratory fibroblasts, and invading cancer cells.
WO 02/20825 relates to novel methods and compositions for detection and isolation of cancer cells with metastatic potential. WO 02/20825 further relates to assays for measuring the metastatic potential of such cancer cells and drug screening assays for the indentification of agents having anti-metastatic potential. Also disclosed are methods and compositions for inhibiting the metastatic potential of cancer cells by modulating the activity of serine integral membrane proteases [(SIMP) consisting of seprase and dipeptidyl peptidase IV (DPIV)] expressed on the surface of metastasizing cancer cells, by using antibodies against SIMP.
Current Treatments of Cancer and Tumor Cell Adhesion
Current cancer treatment regimens comprise surgery, chemotherapy, radiation therapy, and other treatment methods including immunotherapy. Immunotherapy is composed of the usage or the modification of intrinsic bodily mechanisms—in most cases immune mechanisms—to fight cancer. Chemotherapy kills cancer cells through the use of drugs or hormones. Taken either orally or through injection, chemotherapeutic agents are used to treat a wide variety of cancer. They may be given alone or in combination with surgery or radiation or both. Chemotherapy is an established way to destroy hard-to-detect cancer cells that have spread and are circulating in the body. Anemia (low number of red blood cells) is a frequent side effect of chemotherapy and may cause symptoms such as extreme tiredness, dizziness, or shortness of breath. Epoetin alfa (Procrit®, Epogen®)—recombinant erythropoietin that stimulates red blood cell production—is a prescription drug available for the treatment of chemotherapy-related anemia.
Immunotherapy uses the body's own immune system or other parts of the organism to destroy cancer cells. This form of treatment is still being intensively studied in clinical trials; it is not yet widely available to most cancer patients. The various immunological agents used include substances produced by the body (such as the interferons, the interleukins and tumor necrosis factor) and laboratory-produced substances (such as monoclonal antibodies and vaccines). Immunological agents work in different ways and can be used independently or in combination with other forms of treatment.
Angiogenesis Inhibitors as Anti-Metastatic Drugs in Immunotherapy
Angiogenesis inhibitors are drugs that block the development of new blood vessels. Solid tumors cannot grow without inducing the formation of new blood vessels. Blocking the development of new blood vessels cuts off the tumor's supply of oxygen and nutrients.
Several angiogenesis inhibitors are currently being tested in human trials. In cancerous tissue, tumors cannot grow or spread (metastasize) without the development of new blood vessels. Blood vessels supply tissues with oxygen and nutrients necessary for survival and growth.
The present invention provides new uses of DPIV-inhibitors of formulas 1 to 12, and their corresponding pharmaceutically acceptable acid addition salt forms for treating cancer and tumors. In a more preferred embodiment, the compounds of the present invention are useful for the prevention and inhibition of metastasis and tumor colonization.
Reduced expression of the ectopeptidase DPIV and lack of DPIV-like activity in lungs of mutant F344 rats lacking DPIV enzymic activity and expression results in reduced adhesion of cancer cells and in reduced lung metastasis. In vivo cell adhesion and growth of the F344 rat syngeneic mammary adenocarcinoma MADB106 was studied in F344 rats after acutel and chronic treatment with DPIV-ligands in vivo. Mutant F344 substrains lacking DPIV enzymic activity and wild-type-like F344 were tested. Chronic intragastric infusion of isoleucyl cyano pyrrolidine TFA and isoleucyl thiazolidine fumarate via osmotic minipumps over two weeks dose-dependently reduced the cancer-induced weight loss and the number of tumor colonies on the lung surface. Thus, metastasis of MADB106 is reduced by chronic treatment using different DPIV Inhibitors (isoleucyl thiazolidine fumarate; isoleucyl cyano pyrrolidine TFA) suggesting protective-like class effects by the two different DPIV-inhibitor/ligands. Possibly, isoleucyl thiazolidine fumarate and isoleucyl cyano pyrrolidine TFA protect from metastasis either via interaction with cell adhesion processes, via a modification of the cellular host defense mechanisms, via modulation of angiogenesis, via direct effects on cancer cells, or via increased levels of DPIV substrates, which indirectly mediate protective-like effects.
The present invention relates to the area of dipeptidyl peptidase IV (DPIV) inhibition and, more particularly, to a new use of inhibitors of DPIV and DPIV-like enzyme activity for the treatment of cancer and tumors, in particular for the prevention and inhibition of metastasis and tumor colonization, and pharmaceutical compositions containing said compounds.
In contrast to other proposed methods in the art, the present invention provides an orally available therapy with low molecular weight inhibitors of dipeptidyl peptidase IV. The instant invention represents a novel approach for the treatment of cancer and metastatic disease. It is user friendly, commercially useful and suitable for use in a therapeutic regime, especially concerning human diseases.
Spontaneous mutations of the DPIV gene observed in substrains of F344 rats provide a model for studying the role of DPIV in tumor adhesion and colonization. The mutations in F344 rats result in a lack of DPIV enzymatic activity and are found in substrains from Germany (GER) and Japan (JAP) (Thompson et al, 1991; Tsuji et al, 1992), while rats from USA breeders show significant enzyme activity. In F344JAP rats, a G633R substitution in the DPIV protein causes markedly reduced expression of a mutant inactive enzyme (Cheng et al, 1999; Tsuji et al, 1992;), while the other DPIV negative F344GER substrain expresses a non-active mutant enzyme (Thompson et al, 1991). Studies by Pauli and co-workers (Abdel-Ghany et al, 1998; Cheng et al, 1998; Cheng et al, 1999; Johnson et al, 1993) have demonstrated an important role of DPIV/Fibronectin binding in lung metastasis and have discussed the F344JAP rat as a “protein knock-out” model, although this substrain expresses mutant, enzymatically inactive DPIV on endothelial cell surfaces, albeit at greatly reduced levels when compared to expression of wild type DPIV (Cheng et al, 1999).
On the basis of these findings, the investigation of the role of DPIV expression and enzymic activity in cancer and cancer according to the present invention revealed the oral administration of DPIV inhibitors in results in a decrease of lung metastasis and colonization.
The goal of the present invention is the development of dipeptidyl peptidase IV inhibitors and/or ligands, which display a high bioavailability. In another preferred embodiment, the present invention provides DPIV inhibitors, which have an exactly predictable activity time in the target tissue.
Examples for target specific, orally available low molecular weight agents are prodrugs of stable and unstable dipeptidyl peptidase IV inhibitors which comprise general formula A-B-C, whereby A represents an amino acid, B represents the chemical bond between A and C or an amino acid, and C represents an unstable or a stable inhibitor of dipeptidyl peptidase IV respectively. They are described in WO 99/67278, WO 99/67279 the teachings of which are herein incorporated by reference in their entirety.
The present invention relates to a novel method, in which the reduction of activity in the enzyme dipeptidyl peptidase (DPIV or CD 26), or of DPIV-like enzyme activity, or where binding of a DPIV specific ligand exerts tumor suppressive or immunostimulating effects in the organisms of mammals induced by effectors of the enzyme leads as a causal consequence to a reduced growth or adhesion of cancer cells. Such treatment will result in a reduction or delay of cancer cell adhesion (metastasis) or the growth of tumor. As a consequence mammals bearing cancer will benefit from the treatment with inhibitors of DPIV a DPIV-like enzyme activity.
The method of the present invention for treating cancer in an animal, including humans, in need thereof, comprises anti-cancer effects by binding or by inhibiting DPIV, or related enzyme activities, using an inhibitor or ligand of these enzymes. Oral administration of a DPIV inhibitor may be preferable in most circumstances.
The present invention will now be illustrated with reference to the following examples focusing on the anti-cancer-like and anti-metastatic-like action of reduced DPIV-like activity and/or binding in an in vivo cancer cell adhesion assay (example 13), and in cancer colonization assays (example 14).
In one illustrative embodiment, the present invention relates to dipeptide compounds and compounds analogous to dipeptide compounds that are formed from an amino acid and a thiazolidine or pyrrolidine group, and salts thereof, referred to hereinafter as dipeptide compounds.
The use of such compounds as inhibitors of DPIV or of DPIV-analogous enzyme activity is already known from DD 296 075, PCT/DE 97/00820 and PCT/EP 99/03712.
Especially suitable for that purpose according to the invention are dipeptide compounds in which the amino acid is selected from a natural amino acid, such as, for example, leucine, valine, glutamine, glutamic acid, proline, isoleucine, asparagines and aspartic acid.
The dipeptide compounds according to the invention exhibit at a concentration (of dipeptide compounds) of 10 μM, especially under the conditions indicated in Table 1, a reduction in the activity of dipeptidyl peptidase IV or DPIV-analogous enzyme activities of at least 10%, especially of at least 40%. Frequently a reduction in activity of at least 60% or at least 70% is also required. Preferred effectors may also exhibit a reduction in activity of a maximum of 20% or 30%.
Preferred compounds are L-allo-isoleucyl thiazolidine, L-threo-isoleucyl pyrrolidine and salts thereof, especially the fumaric salts, and L-allo-isoleucyl pyrrolidine and salts thereof. Especially preferred compounds are glutaminyl pyrrolidine and glutaminyl thiazolidine of formulas 1 and 2:
In another preferred embodiment, the present invention provides peptide compounds of formula 3 useful for competitive modulation of dipeptidyl peptidase IV catalysis:
A, B, C, D and E are any amino acid residues including proteinogenic amino acids, non-proteinogenic amino acids, L-amino acids and D-amino acids and wherein E and/or D may be absent or B and/or A may be absent with additional conditions as hereinafter detailed:
If B is an amino acid selected from Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic acid, then C is any amino acid residue including D-amino acids, except Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic acid and E may be unused to generate tetrapeptides of the formula A-B-C-D, or D and E may be unused to generate tripeptides of the formula A-B-C provided, but
If B is not an amino acid selected from Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic acid, then C is any α-amino acid except D-amino acids; D is Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic acid; E is any amino acid residue including D-amino acids, except Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic acid, but
If D is Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic acid, then C is any α-amino acid except D-amino acids and Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic acid, and A may be unused to generate tetrapeptides of the formula B-C-D-E, or A and B may be unused to generate tripeptides of the formula C-D-E provided however,
If D is not selected from Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic acid, then E is any amino acid residue including D-amino acids.
Amino acid residues used for the preparation of the compounds of formula (3) can be generally subclassified into four major subclasses as follows.
It is understood, of course, that in a statistical collection of individual residue molecules some molecules will be charged, and some not, and there will be an attraction for or repulsion from an aqueous medium to a greater or lesser extent. To fit the definition of “charged”, a significant percentage (at least approximately 25%) of the individual molecules are charged at physiological pH. The degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary, and, therefore, amino acids specifically contemplated by the invention have been specifically classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behavior.
Amino acid residues can be further subclassified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of 4 carbon atoms or less, inclusive of the carboxyl carbon. Small residues are, of course, always nonaromatic.
For the naturally occurring protein amino acids, subclassification according to the foregoing scheme is as follows.
The gene-encoded amino acid proline, although technically within the group neutral/nonpolar/large/cyclic and nonaromatic, is a special case due to its known effects on the secondary conformation of peptide chains, and is not, therefore, included in this specific defined group.
Certain commonly encountered amino acids, which are not encoded by the genetic code, include, for example, beta-alanine (beta-ala), or other omega-amino acids, such as 3-amino propionic, 4-amino butyric and so forth, alpha-aminoisobutyric acid (Aib), sarcosine (Sar), ornithine (Orn), citrulline (Cit), homoarginine (Har), t-butylalanine (t-butyl-Ala), t-butylglycine (t-butyl-Gly), N-methylisoleucine (N-Melle), phenylglycine (Phg), cyclohexylalanine (Cha), norleucine (Nle), cysteic acid (Cya) and methionine sulfoxide (MSO). These also fall conveniently into particular categories.
Other amino acid substitutions for those encoded in the genetic code can also be included in peptide compounds within the scope of the invention and can be classified within this general scheme.
Proteinogenic amino acids are defined as natural protein-derived α-amino acids. Non-proteinogenic amino acids are defined as all other amino acids, which are not building blocks of common natural proteins.
The resulting peptides may be synthesized as the free C-terminal acid or as the C-terminal amide form. The free acid peptides or the amides may be varied by side chain modifications. Such side chain modifications include for instance, but not restricted to, homoserine formation, pyroglutamic acid formation, disulphide bond formation, deamidation of asparagine or glutamine residues, methylation, t-butylation, t-butyloxycarbonylation, 4-methylbenzylation, thioanysilation, thiocresylation, bencyloxymethylation, 4-nitrophenylation, bencyloxycarbonylation, 2-nitrobencoylation, 2-nitrosulphenylation, 4-toluenesulphonylation, pentafluorophenylation, diphenylmethylation, 2-chlorobenzyloxycarbonylation, 2,4,5-trichlorophenylation, 2-bromobenzyloxycarbonylation, 9-fluorenylmethyloxycarbonylation, triphenylmethylation, 2,2,5,7,8,-pentamethylchroman-6-sulphonylation, hydroxylation, oxidation of methionine, formylation, acetylation, anisylation, bencylation, bencoylation, trifluoroacetylation, carboxylation of aspartic acid or glutamic acid, phosphorylation, sulphation, cysteinylation, glycolysation with pentoses, deoxyhexoses, hexosamines, hexoses or N-acetylhexosamines, farnesylation, myristolysation, biotinylation, palmitoylation, stearoylation, geranylgeranylation, glutathionylation, 5′-adenosylation, ADP-ribosylation, modification with N-glycolylneuraminic acid, N-acetylneuraminic acid, pyridoxal phosphate, lipoic acid, 4′-phosphopantetheine, or N-hydroxysuccinimide.
In the compounds of formula (3), the amino acid residues comprising A, B, C, D, and E substituents are attached to the adjacent moiety according to standard nomenclature so that the amino-terminus (N-terminus) of the amino acids is drawn on the left and the carboxyl-terminus of the amino acid is drawn to the right. Until the present invention by Applicants, known peptide substrates of the proline-specific serine protease dipeptidyl peptidase IV in vitro are the tripeptides Diprotin A (IIe-Pro-IIe), Diprotin B (Val-Pro-Leu) and Diprotin C (Val-Pro-IIe). Applicants have unexpectedly discovered that the compounds disclosed here act as substrates of dipeptidyl peptidase IV in vivo in a mammal and, in pharmacological doses, inhibit the physiological turnover of endogenous substrates by competitive catalysis.
Particularly preferred compounds of the present invention that could be useful as modulators of dipeptidyl peptidase IV and DPIV—like enzymes include those compounds which show Ki-values for DPIV-binding, effectively in DPIV-inhibition in vivo after i.v. and/or p.o. administration to Wistar rats
Further preferred compounds according to the present invention are peptidylketones of formula 4:
Because of the wide distribution of the protein in the body and the wide variety of mechanisms involving DPIV, DPIV-activity and DPIV-related proteins, systemic therapy (enteral or parenteral administration) with DPIV-inhibitors can result in a series of undesirable side-effects.
It has been possible to show that side chain-modified substrates of the enzyme dipeptidyl peptidase IV can be recognised by the enzyme and cleaved in the same way as unmodified substrates (DEMUTH, H.-U., HEINS, J., 1995).
For example, it has been possible to show that phosphorylated dipeptide-(B)-p-nitroanilides [KASPARI, A., et al., 1996] are substrates of DPIV. DPIV-inhibitors such as, for example, Glu(Gly)-Thia or Lys(Z-NO2)-Thia [REINHOLD, D., et al., 1998] are transported completely.
The problem to be solved consisted in preparing compounds that can be used for targeted influencing of locally limited pathophysiological and physiological processes. The problem of the invention especially consists in obtaining locally limited inhibition of DPIV or DPIV-analogous activity for the purpose of targeted intervention in the regulation of the activity of locally active substrates.
This problem is solved according to the invention by providing compounds of the general formula (12)
In accordance with the invention, pharmaceutical compositions are provided comprising at least one compound of the general formula (12)
Throughout the description and the claims for the compounds of formula (12), the expression “alkyl” can denote a C1-50 alkyl group, preferably a C6-30 alkyl group, especially a C8-12 alkyl group; for example, an alkyl group may be a methyl, ethyl, propyl, isopropyl or butyl group. The expression “alk”, for example in the expression “alkoxy”, and the expression “alkan”, for example in the expression “alkanoyl”, are defined as for “alkyl”; aromatic compounds are preferably substituted or optionally unsubstituted phenyl, benzyl, naphthyl, biphenyl or anthracene groups, which preferably have at least 8 C atoms; the expression “alkenyl” can denote a C2-10 alkenyl group, preferably a C2-6 alkenyl group, which has the double bond(s) at any desired location and may be substituted or unsubstituted; the expression “alkynyl” can denote a C2-10 alkynyl group, preferably a C2-6 alkynyl group, which has the triple bond(s) at any desired location and may be substituted or unsubstituted; the expression “substituted” or substituent can denote any desired substitution by one or more, preferably one or two, alkyl, alkenyl, alkynyl, mono- or multi-valent acyl, alkanoyl, alkoxyalkanoyl or alkoxyalkyl groups; the afore-mentioned substituents may in turn have one or more (but preferably zero) alkyl, alkenyl, alkynyl, mono- or multi-valent acyl, alkanoyl, alkoxyalkanoyl or alkoxyalkyl groups as side groups; organic amines, amides, alcohols or acids, each having from 8 to 50 C atoms, preferably from 10 to 20 C atoms, can have the formulae (alkyl)2N— or alkyl-NH—, —CO—N(alkyl)2 or —CO—NH(alkyl), -alkyl-OH or -alkyl-COOH.
Despite an extended side chain function, the compounds of formula (12) can still bind to the active centre of the enzyme dipeptidyl peptidase IV and analogous enzymes but are no longer actively transported by the peptide transporter PepT1. The resulting reduced or greatly restricted transportability of the compounds according to the invention leads, in ideal manner, to local or site directed inhibition of DPIV and DPIV-like enzyme activity.
The compounds of formula (12) or compounds used in accordance with the invention can be present or used, respectively, in the form of racemates or in the form of enantiomerically pure compounds, preferably in the L-threo or L-allo form with respect to part A of formula (12).
By extending/expanding the side chain modifications, for example beyond a number of seven carbon atoms, it is accordingly possible to obtain a dramatic reduction in transportability (see Example 12). The Examples in Table 12.1 clearly show that, with increasing spatial size of the side chains, there is a reduction in the transportability of the substances. By spatially and sterically expanding the side chains, for example beyond the atom group size of a monosubstituted phenyl radical, hydroxylamine radical or amino acid residue, it is possible according to the invention to modify or suppress the transportability of the target substances.
According to the present invention, the compounds of formula (12) inhibit DPIV or DPIV-like enzyme activity in the body of a mammal in a site specific manner. It is accordingly possible to influence local physiological and pathophysiological conditions (inflammation, psoriasis, arthritis, autoimmune diseases, allergies) effectively and with dramatically reduced side-effects.
Preferred compounds of formula (12) are compounds, wherein the oligopeptides have chain lengths of from 3 to 15, especially from 4 to 10, amino acids, and/or the polyethylene glycols have molar masses of at least 250 g/mol, preferably of at least 1500 g/mol and up to 15 000 g/mol, and/or the optionally substituted organic amines, amides, alcohols, acids or aromatic compounds have at least 12 C atoms and preferably up to 30 C atoms.
The compounds of the present invention can be converted into acid addition salts, especially pharmaceutically acceptable acid addition salts. The pharmaceutically acceptable salt generally takes a form in which an amino acids basic side chain is protonated with an inorganic or organic acid. Representative organic or inorganic acids include hydrochloric, hydrobromic, perchloric, sulfuric, nitric, phosphoric, acetic, propionic, glycolic, lactic, succinic, maleic, fumaric, malic, tartaric, citric, benzoic, mandelic, methanesulfonic, hydroxyethanesulfonic, benzenesulfonic, oxalic, pamoic, 2-naphthalenesulfonic, p-toulenesulfonic, cyclohexanesulfamic, salicylic, saccharinic or trifluoroacetic acid. All pharmaceutically acceptable acid addition salt forms of the compounds of formulas (1) to (12) are intended to be embraced by the scope of this invention.
In view of the close relationship between the free compounds and the compounds in the form of their salts, whenever a compound is referred to in this context, a corresponding salt is also intended, provided such is possible or appropriate under the circumstances.
The present invention further includes within its scope prodrugs of the compounds of this invention. In general, such prodrugs will be functional derivatives of the compounds which are readily convertible in vivo into the desired therapeutically active compound. Thus, in these cases, the methods of treatment of the present invention, the term “administering” shall encompass the treatment of the various disorders described with prodrug versions of one or more of the claimed compounds, but which converts to the above specified compound in vivo after administration to the subject. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in “Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985 and the patent applications DE 198 28 113 and DE 198 28 114, which are fully incorporated herein by reference.
Where the compounds according to this invention have at least one chiral center, they may accordingly exist as enantiomers. Where the compounds possess two or more chiral centers, they may additionally exist as diastereomers. It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention. Furthermore, some of the crystalline forms of the compounds may exist as polymorphs and as such are intended to be included in the present invention. In addition, some of the compounds may form solvates with water (i.e. hydrates) or common organic solvents, and such solvates are also intended to be encompassed within the scope of this invention.
The compounds, including their salts, can also be obtained in the form of their hydrates, or include other solvents used for their crystallization.
As indicated above, the compounds of the present invention, and their corresponding pharmaceutically acceptable acid addition salt forms, are useful in inhibiting DPIV and DPIV—like enzyme activity. The ability of the compounds of the present invention, and their corresponding pharmaceutically acceptable acid addition salt forms to inhibit DPIV and DPIV—like enzyme activity may be demonstrated employing the DPIV activity assay for determination of the Ki-values and the IC50-values in vitro, as described in examples 7 and 8.
The ability of the compounds of the present invention, and their corresponding pharmaceutically acceptable acid addition salt forms to inhibit DPIV in vivo may be demonstrated by oral or intravasal administration to Wistar rats, as described in example 11. The compounds of the present invention inhibit DPIV activity in vivo after both, oral and intravasal administration to Wistar rats.
DPIV is present in a wide variety of mammalian organs and tissues e.g. the intestinal brush-border (Gutschmidt S. et al., “In situ”—measurements of protein contents in the brush border region along rat jejunal villi and their correlations with four enzyme activities. Histochemistry 1981, 72 (3), 467–79), exocrine epithelia, hepatocytes, renal tubuli, endothelia, myofibroblasts (Feller A. C. et al., A monoclonal antibody detecting dipeptidylpeptidase IV in human tissue. Virchows Arch. A. Pathol. Anat. Histopathol. 1986; 409 (2):263–73), nerve cells, lateral membranes of certain surface epithelia, e.g. Fallopian tube, uterus and vesicular gland, in the luminal cytoplasm of e.g., vesicular gland epithelium, and in mucous cells of Brunner's gland (Hartel S. et al., Dipeptidyl peptidase (DPP) IV in rat organs. Comparison of immunohistochemistry and activity histochemistry. Histochemistry 1988; 89 (2): 151–61), reproductive organs, e.g. cauda epididymis and ampulla, seminal vesicles and their secretions (Agrawal & Vanha-Perttula, Dipeptidyl peptidases in bovine reproductive organs and secretions. Int. J. Androl. 1986, 9 (6): 435–52). In human serum, two molecular forms of dipeptidyl peptidase are present (Krepela E. et al., Demonstration of two molecular forms of dipeptidyl peptidase IV in normal human serum. Physiol. Bohemoslov. 1983, 32 (6): 486–96). The serum high molecular weight form of DPIV is expressed on the surface of activated T cells (Duke-Cohan J. S. et al., Serum high molecular weight dipeptidyl peptidase IV (CD26) is similar to a novel antigen DPPT-L released from activated T cells. J. Immunol. 1996, 156 (5): 1714–21).
The compounds of the present invention, and their corresponding pharmaceutically acceptable acid addition salt forms are able to inhibit DPIV in vivo. In one embodiment of the present invention, all molecular forms, homologues and epitopes of DPIV from all mammalian tissues and organs, also of those, which are undiscovered yet, are intended to be embraced by the scope of this invention.
Among the rare group of proline-specific proteases, DPIV was originally believed to be the only membrane-bound enzyme specific for proline as the penultimate residue at the amino-terminus of the polypeptide chain. However, other molecules, even structurally non-homologous with the DPIV but bearing corresponding enzyme activity, have been identified recently. DPIV-like enzymes, which are identified so far, are e.g. fibroblast activation protein α, dipeptidyl peptidase IV β, dipeptidyl aminopeptidase-like protein, N-acetylated α-linked acidic dipeptidase, quiescent cell proline dipeptidase, dipeptidyl peptidase II, attractin and dipeptidyl peptidase IV related protein (DPP 8), and are described in the review article by Sedo & Malik (Sedo & Malik, Dipeptidyl peptidase IV-like molecules: homologous proteins or homologous activities? Biochimica et Biophysica Acta 2001, 36506: 1–10). Further DPIV like enzymes are disclosed in WO 01/19866, WO 02/04610 and WO 02/34900. WO 01/19866 discloses novel human dipeptidyl aminopeptidase (DPP8) with structural und functional similarities to DPIV and fibroblast activation protein (FAP). The dipeptidyl peptidase IV-like enzyme of WO 02/04610 is well known in the art. In the Gene Bank data base, this enzyme is registered as KIAA1492. In another preferred embodiment of the present invention, all molecular forms, homologues and epitopes of proteins comprising DPIV-like enzyme activity, from all mammalian tissues and organs, also of those, which are undiscovered yet, are intended to be embraced by the scope of this invention.
The ability of the compounds of the present invention, and their corresponding pharmaceutically acceptable acid addition salt forms to inhibit DPIV-like enzymes may be demonstrated employing an enzyme activity assay for determination of the Ki-values in vitro as described in example 9. The Ki-values of the compounds of the present invention against porcine dipeptidyl peptidase II were exemplary determined as Ki=8.52*10−5 M±6.33*10−6 M for glutaminyl pyrrolidine and Ki=1.07*10−5 M±3.81*10−7 M for glutaminyl thiazolidine.
In another embodiment, the compounds of the present invention, and their corresponding pharmaceutically acceptable acid addition salt forms have only low, if no inhibitory activity against non-DPIV and non-DPIV-like proline specific enzymes. As described in example 10, with glutaminyl thiazolidine and glutaminyl pyrrolidine exemplarily, no inhibition of dipeptidyl peptidase I and prolyl oligopeptidase was found. Against prolidase, both compounds explained a marked lower efficacy compared to DPIV. The IC 50-values against prolidase were determined as IC 50>3 mM for glutaminyl thiazolidine and as IC 50=3.4*10−4M±5.63*10−5 for glutaminyl pyrrolidine.
The present invention provides a method of treating a condition mediated by modulation of the DPIV or DPIV-like enzyme activity in a subject in need thereof which comprises administering any of the compounds of the present invention or pharmaceutical compositions thereof in a quantity and dosing regimen therapeutically effective to treat the condition. Additionally, the present invention includes the use of the compounds of this invention, and their corresponding pharmaceutically acceptable acid addition salt forms, for the preparation of a medicament for the treatment of a condition mediated by modulation of the DPIV activity in a subject. The compound may be administered to a patient by any conventional route of administration, including, but not limited to, intravenous, oral, subcutaneous, intramuscular, intradermal and parenteral.
In a further illustrative embodiment, the present invention provides formulations for the compounds of formulas 1 to 12, and their corresponding pharmaceutically acceptable acid addition salt forms, in pharmaceutical compositions.
The term “subject” as used herein, refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.
The term “therapeutically effective amount” as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human, being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
As used herein, the term “composition” is intended to encompass a product comprising the claimed compounds in the therapeutically effective amounts, as well as any product which results, directly or indirectly, from combinations of the claimed compounds.
To prepare the pharmaceutical compositions of this invention, one or more compounds of formulas 1 to 12, and their corresponding pharmaceutically acceptable acid addition salt forms, as the active ingredient, is intimately admixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques, which carrier may take a wide variety of forms depending of the form of preparation desired for administration, e.g., oral or parenteral such as intramuscular. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed. Thus, for liquid oral preparations, such as for example, suspensions, elixirs and solutions, suitable carriers and additives may advantageously include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like; for solid oral preparations such as, for example, powders, capsules, gelcaps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are employed. If desired, tablets may be sugar coated or enteric coated by standard techniques. For parenterals the carrier will usually comprise sterile water, through other ingredients, for example, for purposes such as aiding solubility or for preservation, may be included.
Injectable suspensions may also prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. The pharmaceutical compositions herein will contain, per dosage unit, e.g., tablet, capsule, powder, injection, teaspoonful and the like, an amount of the active ingredient necessary to deliver an effective dose as described above. The pharmaceutical compositions herein will contain, per unit a dosage unit, e.g., tablet, capsule, powder, injection, suppository, teaspoonful and the like, of from about 0.03 mg to 100 mg/kg (preferably 0.1–30 mg/kg) and may be given at a dosage of from about 0.1–300 mg/kg per day (preferably 1–50 mg/kg per day). The dosages, however, may be varied depending upon the requirement of the patients, the severity of the condition being treated and the compound being employed. The use of either daily administration or post-periodic dosing may be employed. Typically the dosage will be regulated by the physician based on the characteristics of the patient, his/her condition and the therapeutic effect desired.
Preferably these compositions are in unit dosage forms from such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampoules, autoinjector devices or suppositories; for oral parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. Alternatively, the composition may be presented in a form suitable for once-weekly or once-monthly administration; for example, an insoluble salt of the active compound, such as the decanoate salt, may be adapted to provide a depot preparation for intramuscular injection. For preparing solid compositions such as tablets, the principal active ingredient is ideally mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is ideally dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective dosage forms such as tablets, pills and capsules. This solid preformulation composition may then be subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention.
The tablets or pills of the novel composition can be advantageously coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
This liquid forms in which the novel compositions of the present invention may be advantageously incorporated for administration orally or by injection include, aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or gelatin.
Where the processes for the preparation of the compounds according to the invention give rise to mixture of stereoisomers, these isomers may be separated by conventional techniques such as preparative chromatography. The compounds may be prepared in racemic form, or individual enantiomers may be prepared either by enantiospecific synthesis or by resolution. The compounds may, for example, be resolved into their components enantiomers by standard techniques, such as the formation of diastereomeric pairs by salt formation with an optically active acid, such as (−)-di-p-toluoyl-d-tartaric acid and/or (+)-di-p-toluoyl-l-tartaric acid followed by fractional crystallization and regeneration of the free base. The compounds may also resolved by formation of diastereomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compounds may be resolved using a chiral HPLC column.
During any of the processes for preparation of the compounds of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W. Greene & P. G. M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, fully incorporated herein by reference. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.
The method of treating conditions modulated by dipeptidyl peptidase IV and DPIV-like enzymes described in the present invention may also be carried out using a pharmaceutical composition comprising any of the compounds as defined herein and a pharmaceutically acceptable carrier. The pharmaceutical composition may contain between about 0.01 mg and 1000 mg, preferably about 5 to 500 mg, of the compound, and may be constituted into any form suitable for the mode of administration selected. Carriers include necessary and inert pharmaceutical excipients, including, but not limited to, binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, dyes, and coatings. Compositions suitable for oral administration include solid forms, such as pills, tablets, caplets, capsules (each including immediate release, timed release and sustained release formulations), granules, and powders, and liquid forms, such as solutions, syrups, elixirs, emulsions, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions and suspensions.
Advantageously, compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, compounds of the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen and dosage strength will need to be accordingly modified to obtain the desired therapeutic effects.
More preferably, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders; lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or betalactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and other compounds known within the art.
The liquid forms are suitable in flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired.
The compound of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines using processes well described in the art.
Compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol, or polyethyl eneoxidepolyllysine substituted with palmitoyl residue. Furthermore, compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polyactic acid, polyepsilon caprolactone, polyhydroxy butyeric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
Compounds of this invention may be administered in any of the foregoing compositions and according to dosage regimens established in the art whenever treatment of the addressed disorders is required.
The daily dosage of the products may be varied over a wide range from 0.01 to 1.000 mg per adult human per day. For oral administration, the compositions are preferably provided in the form of tablets containing, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150, 200, 250 and 500 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.1 mg/kg to about 300 mg/kg of body weight per day. Preferably, the range is from about 1 to about 50 mg/kg of body weight per day. The compounds may be administered on a regimen of 1 to 4 times per day.
Optimal dosages to be administered may be readily determined by those skilled in the art, and will vary with the particular compound used, the mode of administration, the strength of the preparation, bioavailability due to the mode of administration, and the advancement of disease condition. In addition, factors associated with the particular patient being treated, including patient age, weight, diet and time of administration, should generally be considered in adjusting dosages.
1.1 General Synthesis of Isoleucyl Thiazolidine Salt
The Boc-protected amino acid BOC-Ile-OH is placed in ethyl acetate and the batch is cooled to about −5° C. N-Methylmorpholine is added dropwise, pivalic acid chloride (on a laboratory scale) or neohexanoyl chloride (on a pilot-plant scale) is added dropwise at constant temperature. The reaction is stirred for a few minutes for activation. N-Methylmorpholine (laboratory scale) and thiazolidine hydrochloride (laboratory scale) are added dropwise in succession, thiazolidine (pilot-plant scale) is added. Working-up in the laboratory is effected in conventional manner using salt solutions, on a pilot-plant scale the batch is purified with NaOH and CH3COOH solutions.
The removal of the BOC protecting group is carried out using HCl/dioxane (laboratory scale) or H2SO4 (pilot-plant scale). In the laboratory the hydrochloride is crystallised from EtOH/ether.
On a pilot-plant scale the free amine is prepared by the addition of NaOH/NH3. Fumaric acid is dissolved in hot ethanol, the free amine is added dropwise, and (IIe-Thia)2 furmarate (M=520.71 gmol31 1) precipitates. The analysis of isomers and enantiomers is carried out by electrophoresis.
1.2 Synthesis of Glutaminyl Pyrrolidine Free Base
N-Benzyl-oxycarbonylglutamine (2.02 g, 7.21 mmol) was dissolved in 35 ml THF and brought to −15° C. Into that mixture CAIBE (isobutylchloroformiate) (0.937 ml, 7.21 mmol) and 4-methylmorpholine (0.795 ml, 7.21 mmol) where added and the solution was stirred for 15 min. The formation of the mixed anhydride was checked by TLC (eluent: CHCl3/MeOH: 9/1). After warming to −10° C. pyrrolidine (0.596 ml, 7.21 mmol) was added. The mixture was brought to room temperature and stirred overnight.
The sediment formed was filtered off and the solvent was evaporated. The resulting oil was taken up in ethylacetate (20 ml) and washed with a saturated solution of sodiumhydrogensulfate followed by a saturated solution of sodiumbicarbonate, water and brine. The organic layer was separated, dried and evaporated. The resulting product was checked for purity by TLC (eluent: CHCl3/MeOH: 9/1).
Yield: 1.18 g, waxy solid.
1.18 g of the resulting solid Z-protected compound was dissolved in 40 ml absolute ethanol. Into the solution ca. 20 mg Pd on charcoal (10%, FLUKA) was added and the suspension was shaken under a hydrogen atmosphere for 3 h. The progress of the reaction was monitored by TLC (eluent: CHCl3/MeOH: 9/1). After completion of the reaction the was removed to provide the free base.
Yield: 99%. The purity was checked by means of TLC: n-butanole/AcOH/water/ethylacetate: 1/1/1/1, Rf=0.4. The identity of the reaction product was checked by NMR analysis.
1.3 Synthesis of Glutaminyl Thiazolidine Hydrochloride
N-t-Butyl-oxycarbonylglutamine (2.0 g, 8.12 mmol) was dissolved in 5 ml THF and brought to −15° C. Into that mixture CAIBE (isobutylchloroformiate) (1.06 ml, 8.12 mmol) and 4-methylmorpholine (0.895 ml, 8.12 mmol) where added and the solution was stirred for 15 min. The formation of the mixed anhydride was checked by TLC (eluent: CHCl3/MeOH: 9/1). After warming to −10° C. another equivalent 4-methylmorpholine (0.895 ml, 8.12 mmol) and thiazolidinehydrochloride (1.02 g, 8.12 mmol was added. The mixture was brought to room temperature and stirred overnight.
The sediment formed was filtered off and the solvent was evaporated. The resulting oil was taken up in chloroform (20 ml) and washed with a saturated solution of sodiumhydrogensulfate followed by a saturated solution of sodiumbicarbonate, water and brine. The organic layer was separated, dried and evaporated. The resulting product was checked for purity by TLC (eluent: CHCl3/MeOH: 9/1).
Yield: 1.64 g, solid.
640 mg of the resulting solid Boc-protected compound was dissolved in 3.1 ml ice cold HCl in dioxane (12.98 M, 20 equivalents) and left on ice. The progress of the reaction was monitored by TLC (eluent: CHCl3/MeOH: 9/1). After completion of the reaction the solvent was removed and the resulting oil was taken up in methanole and evaporated again. After that the resulting oil was dried over phosphorous-V-oxide and triturated two times with diethylether. The purity was checked by HPLC.
Yield: 0.265 g. The purity was checked by HPLC. The identity of the reaction product was checked by NMR analysis.
1.4 Synthesis of Glutaminyl Pyrrolidine Hydrochloride
N-t-Butyl-oxycarbonylglutamine (3.0 g, 12.18 mmol) was dissolved in 7 ml THF and brought to −15° C. Into that mixture CAIBE (isobutylchloroformiate) (1.6 ml, 12.18 mmol) and 4-methylmorpholine (1.3 ml, 12.18 mmol) where added and the solution was stirred for 15 min. The formation of the mixed anhydride was checked by TLC (eluent: CHCl3/MeOH: 9/1). After warming to −10° C. 1 equivalent of pyrrolidine (1.0 ml, 12.18 mmol) was added. The mixture was brought to room temperature and stirred overnight.
The sediment formed was filtered off and the solvent was evaporated. The resulting oil was taken up in chloroform (20 ml) and washed with a saturated solution of sodiumhydrogensulfate followed by a saturated solution of sodiumbicarbonate, water and brine. The organic layer was separated, dried and evaporated. The resulting product was checked for purity by TLC (eluent: CHCl3/MeOH: 9/1).
Yield: 2.7 g solid.
2.7 g of the resulting solid was dissolved in 13.0 ml ice cold HCl in dioxane (12.98 M, 20 equivalents) and left on ice. The progress of the reaction was monitored by TLC (eluent: CHCl3/MeOH: 9/1). After completion of the reaction the solvent was removed and the resulting oil was taken up in methanole and evaporated again. After that the resulting oil was dried over phosphorous-V-oxide and triturated two times with diethylether.
Yield: 980 mg. The purity was checked by HPLC. The identity of the reaction product was checked by NMR analysis.
2.1 Melting Point Determination
Melting points were determined on a Kofler heating platform microscope from Leica Aktiengesellschaft, the values are not corrected, or on a DSC apparatus (Heumann-Pharma).
2.2 Optical Rotation
The rotation values were recorded at different wavelengths on a “Polarimeter 341” or higher, from the Perkin-Elmer company.
2.3 Measurement Conditions for the Mass Spectroscopy
The mass spectra were recorded by means of electrospray ionisation (ESI) on an “API 165” or “API 365” from the PE Sciex company. The operation is carried out using an approximate concentration of c=10 μg/ml, the substance is taken up in MeOH/H2O 50:50, 0.1% HCO2H, the infusion is effected using a spray pump (20 μl/min). The measurement were made in positive mode [M+H]+, the ESI voltage is U=5600V.
2.4.1 Tests on Isoleucyl Thiazolidine Fumarate (Isomer)
The NMR and HPLC data confirm the identity of the substance in question.
2.4.2 Tests on Other Isoleucyl Thiazolidine Salts
All syntheses were carried out on a peptide synthesizer SP 650 (Labortec AG) applying Fmoc/tBu-strategy. Protected amino acids were purchased from Novabiochem or Bachem. trifluoro acetic acid (TFA) was purchased from Merck, triisopropyl silane (TIS) was purchased from Fluka.
Pre-loaded Fmoc-Yaa-Wang resin (2.8 g/substitution level 0.57 mmol/g) was deprotected using 20% piperidine/N,N-dimethylformamide (DMF). After washing with DMF a solution of 2 eq (1.1 g) of Fmoc-Pro-OH were solved in DMF (12 ml solvent per gram resin). 2 eq (1.04 g) of 2-(1H-Benzotriazole 1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and 4 eq (1.11 ml) of N,N-diisopropylethylamine (DIEA) were added and placed in the reaction vessel. The mixture was shaken at room temperature for 20 minutes. Then the coupling cycle was repeated. After subsequent washing with DMF, dichlormethane, isopropanol and diethyl ether the resulting Fmoc-Pro-IIe-Wang resin was dried and then divided into 6 parts before coupling the last amino acid derivative.
Fmoc protecting group was removed as described above. After that 0.54 mmol of the Boc-amino acid, 0.54 mmol TBTU and 0.108 mmol DIEA in DMF were shaken for 20 min. The coupling cycle was repeated. Finally the peptide resin was washed and dried described above.
The peptide was cleaved from the resin using a mixture of trifluoroacetic acid (TFA) for 2.5 h, containing the following scavengers: TFA/H2O/triisipropylsilane (TIS)=9.5/0.25/0.25
The yields of crude peptides were 80–90% on the average. The crude peptide was purified by HPLC on a Nucleosil C18 column (7 μm, 250*21.20 mm, 100 A) using a linear gradient of 0.1% TFA/H2O with increasing concentration of 0.1% TFA/acetonitrile (from 5% to 65% in 40 min) at 6 ml/min.
The pure peptide was obtained by lyophilization, identified by Electrospray mass spectrometry and HPLC analysis.
3.1 Results—Identification of Xaa-Pro-Yaa Tripeptides After Chemical Synthesis
Boc-Val-OH (3.00 g, 13.8 mmol) was dissolved in 10 ml of dry THF and cooled down to −15° C. To the mixture CAIBE (1.80 ml, 13.8 mmol) and NMM (1.52 ml, 13.8 mmol) where added and the solution was stirred until the formation of the mixed anhydride was complete. Then the mixture was brought to −10° C. and NMM (1.52 ml, 13.8 mmol) was added followed by H-Pro-OMe*HCl (2.29 g, 13.8 mmol). The mixture was allowed to reach room temperature and left overnight. After removing the solvent and the usual workup the resulting ester 1 was taken without further characterisation. The ester 1 was dissolved in HCl/HOAc (5 ml, 6N) and left at 0° C. until the removal of the Boc-group was complete. The solvent was then removed and the resulting oil was treated with diethylether to give a white solid 2.
Yield: 2.5 g, 80%. Z-Ala-Val-Pro-OMe 3
Z-Ala OH (3.5 g, 15.7 mmol) and 2 (4.18 g, 15.7 mmol) where treated in the same manner as above for 1, to give 3 as a white solid.
Yield: 4.2 g, 64%. Z-Ala-Val-Pro-OH 4
3 (4.2 g, 9.6 mmol) was dissolved in 30 ml of water/acetone (1/5 v/v) and 11.6 ml NaOH (1N) where added. After completion of the reaction the organic solvent was removed by evaporation and the resulting solution was diluted by 15 ml NaHCO3 solution (saturated). Then the mixture was extracted three times by 10 ml of acetic acid ethyl ester. After that the solution was brought to pH2 by adding HCl (15% in water). The resulting mixture was extracted three times by 30 ml of acetic acid ethyl ester. The organic layer was separated and washed three times with brine, dried (Na2SO4) and evaporated.
Yield: 3.5 g, 87%. Z-Ala-Val-Pro-CH2—Br 5
4 (2.00 g, 4.76 mmol) was dissolved in 15 ml of dry THF and converted into a mixed anhydride (see compound 1) using CAIBE (0.623 ml, 4.76 mmol) and NMM (0.525 ml, 4.76 mmol). The precipitate formed was filtered off and cooled down to −15° C. Then diazomethane (23.8 mmol in 30 ml ether) was dropped into the solution under an argon atmosphere. After leaving the mixture for 1 h at 0° C. 1.27 ml of HBr (33% in AcOH) was added and the solution was stirred for 30 min at room temperature. After that 70 ml of ether was added and the mixture was washed with 20 ml of water. The organic layer was separated and dried (Na2SO4) and evaporated.
Yield (crude): 1.8 g, 80%. Z-protected acyloxymethylene ketones
The acid (2 eq) was dissolved in DMF and an equimolar amount of KF was added. The suspension was allowed to stir at room temperature for 1 hour. Then the brommethylene (1 eq) component was added and the solution was allowed to stir overnight. After that the solvent was removed under vacuum and the resulting oil was dissolved in chloroform and washed with brine. Then the organic layer was separated dried (Na2SO4) and the solvent was removed. The product was purified by column chromatography using silica gel and heptane/chloroform.
Yield: 0.351 g, 36% Z-Ala-Val-Pro-CH2O—C(O)-Ph 7
Yield: 0.34 g, 56% Deprotection
The Z-protected compound was dissolved in HBr/AcOH and stirred. When the reaction was complete ether was added, the white precipitate formed was filtered off and dried.
Yield: 0.252 g, 98% H-Ala-Val-Pro-CH2O—C(O)Ph*HBr 9
Yield: 0.251 g, 99%
Oxalylchloride (714 μl, 8.28 mmol) was dissolved in 10 ml of dry dichlormethane and brought to −78° C. Then DMSO (817 μl, 8.28 mmol) was added dropwise. The solution was stirred for 20 min at −78° C. Then 1 (1.00 g, 4.6 mmol) was added and the mixture was stirred for 20 min. After that TEA (2.58 ml, 18.4 mmol) was added and the mixture was allowed to reach room temperature. The mixture was diluted with hexane/ethylacetate (2/1 v/v) and 10 ml of HCl (10% in water) was added. The organic layer was separated and the aqueous phase was extracted with 20 ml of methylenechloride. All organic layers were collected and washed with brine, followed by water, then dried. The product was purified by column chromatography using silica gel and heptane/chloroform.
Yield: 0.52 g, 52% tert-butyl N-1-[cyclopentyl(hydroxy)methyl]-2-methylbutylcarbamate 3
2 (0.52 g, 2.42 mmol) was dissolved in 10 ml of dry THF and cooled down to 0° C. Then cyclopentylmagnesiumbromide (1.45 ml of a 2 M solution) was added. After completion of the reaction (2 ml) of water was added and solution was neutralized by adding aqueous HCl. Then methylenechloride was added and the organic layer was separated and dried (Na2SO4). After evaporation the resulting oil was used without further characterisation.
tert-butyl N-[1-(cyclopentylcarbonyl)-2-methylbutyl]carbamate 4
3 (0.61 g, 2.15 mmol) was treated like 1. Oxalylchloride (333 μl, 3.87 mmol), DMSO (382 μl, 5.37 mmol), TEA (1.2 ml, 8.59 mmol)
Yield: 0.180 g, 30% 1-cyclopentyl-3-methyl-1-oxo-2-pentanaminium chloride 5
4 (0.18 g, 0.63 mmol) was dissolved in 2 ml HCl (7 N in dioxane). After completion of the reaction the solvent was removed and the resulting oil was purified by column chromatography on silical gel using a chloroform/methanol/water gradient. The resulting oil was triturated with ether.
Yield: 0.060 g, 54%
6.1 Synthesis of Boc-glutamyl-thiazolidine (Boc-Glu-Thia)
Reaction of Boc-Glu(OMe)-OH with Thia*HCl according to Method B (see section 6.4 for methods), hydrolysis of Boc-Glu(OMe)-Thia according to Method G
6.1.1 Analytical Data for Boc-Glu-Thia
Boc-Glu-Thia was modified at the γ-carboxylic acid function by introducing radicals of varying size. The radicals were coupled by way of their amino group by forming an amide bond to the γ-carboxylic acid function, with a variety of coupling methods being used depending on the radical. The following amino components were attached to Boc-Glu-Thia using the method stated:
In 2 cases, purification of the reaction products differs from the general description of synthesis.
The product already precipitates out from the mixture on stirring overnight; it is subsequently filtered off and washed with 0.1N HCl and copious amounts of water and then dried over P4O10 in vacuo.
In contrast to the general procedure, the starting materials for the synthesis are dissolved in a 500-fold excess of DMF. After the reaction is complete, the DMF is completely removed in vacuo and the residue is dissolved in a large amount of methanol. After ether is poured on, to form an upper layer, the product precipitates out together with the unreacted PEG. Fine purification was carried out by preparative HPLC separation on a gel filtration column (Pharmazia, Sephadex G-25, 90 μm, 260 mm–100 mm).
Separating conditions: eluant: water; flow rate: 5 ml/min; λ=220 nm
6.2.2 Synthesis Data for Side Chain-Modified Boc-glutamyl Thiazolidines
The N-terminal Boc protecting groups were cleaved off the compounds described in Table 6.2.2 using method F. The substances modified with Gly derivatives were purified by preparative HPLC separation and are present as trifluoroacetates. The H-Glu(PEG)-Thia was purified on a gel filtration column in the same manner as the Boc-protected precursor.
6.3.1 Synthesis Data for Side Chain-Modified Glutamyl Thiazolidines
10 mmol of N-terminally protected amino acid or peptide are dissolved in 20 ml of absolute THF. The solution is cooled to −15° C.±2° C. With stirring in each case, 10 mmol of N-MM and 10 mmol of chloroformic acid isobutyl ester are added in succession, the stated temperature range being strictly adhered to. After approximately 6 min, 10 mmol of the amino component is added. When the amino component is a salt, a further 10 mmol of N-MM is then added to the reaction mixture. The reaction mixture is then stirred for 2 h in the cold state and overnight at room temperature.
The reaction mixture is concentrated using a rotary evaporator, taken up in EA, washed with 5% KH2SO4 solution, saturated NaHCO3 solution and saturated NaCl solution and dried over NaSO4. After removal of the solvent in vacuo, the compound is recrystallized from EA/pentane.
Method B: Peptide Bond Attachment by the Mixed Anhydride Method using Pivalic Acid Chloride as Activation Reagent
10 mmol of N-terminally protected amino acid or peptide are dissolved in 20 ml of absolute THF. The solution is cooled to 0° C. With stirring in each case, 10 mmol of N-MM and 10 mmol of pivalic acid chloride are added in succession, the stated temperature range being strictly adhered to. After approximately 6 min, the mixture is cooled to −15° C. and, once the lower temperature has been reached, 10 mmol of the amino component is added. When the amino component is a salt, a further 10 mmol of N-MM is then added to the reaction mixture. The reaction mixture is then stirred for 2 h in the cold state and overnight at room temperature.
Further working up is carried out as in Method A.
Method C: Peptide Bond Attachment Using TBTU as Activation Reagent
10 mmol of the N-terminally protected amino acid or peptide and 10 mmol of the C-terminally protected amino component are dissolved in 20 ml of absolute DMF. The solution is cooled to 0° C. With stirring in each case, 10 mmol of DIPEA and 10 mmol of TBTU are added in succession. The reaction mixture is stirred for one hour at 0° C. and then overnight at room temperature. The DMF is completely removed in vacuo and the product is worked up as described in Method A.
Method D: Synthesis of an Active Ester (N-hydroxysuccinimide Ester)
10 mmol of N-terminally protected amino acid or peptide and 10 mmol of N-hydroxysuccinimide are dissolved in 20 ml of absolute THF. The solution is cooled to 0° C. and 10 mmol of dicyclohexylcarbodiimide are added, with stirring. The reaction mixture is stirred for a further 2 h at 0° C. and then overnight at room temperature. The resulting N,N′-dicyclohexylurea is filtered off and the solvent is removed in vacuo and the remaining product is recrystallized from EA/pentane.
Method E: Amide Bond Attachment Using N-hydroxysuccinimide Esters
10 mmol of the C-terminally unprotected amino component is introduced into an NaHCO3 solution (20 mmol in 20 ml of water). At room temperature and with stirring, 10 mmol of the N-terminally protected N-hydroxysuccinimide ester dissolved in 10 ml of dioxane are slowly added dropwise. Stirring of the reaction mixture is continued overnight and the solvent is then removed in vacuo.
Further working up is carried out as in Method A.
Method F: Cleavage of the Boc Protecting Group
3 ml of 1.1N HCl/glacial acetic acid (Method F1) or 3 ml of 1.1N HCl/dioxane (Method F2) or 3 ml of 50% TFA in DCM (Method F3) are added to 1 mmol of Boc-protected amino acid pyrrolidide, thiazolidide or peptide. The cleavage at RT is monitored by means of TLC. After the reaction is complete (approximately 2 h), the compound is precipitated in the form of the hydrochloride using absolute diethyl ether and is isolated with suction and dried over P4O10 in vacuo. Using methanol/ether, the product is recrystallized or reprecipitated.
Method G: Hydrolysis
1 mmol of peptide methyl ester is dissolved in 10 ml of acetone and 11 ml of 0.1M NaOH solution and stirred at room temperature. The course of the hydrolysis is monitored by means of TLC. After the reaction is complete, the acetone is removed in vacuo. The remaining aqueous solution is acidified, using concentrated KH2SO4 solution, until a pH of 2–3 is reached. The product is then extracted several times using EA; the combined ethyl acetate fractions are washed with saturated NaCl solution and dried over NaSO4, and the solvent is removed in vacuo. Crystallization from EA/pentane is carried out.
For Ki determination, dipeptidyl peptidase IV from porcine kidney with a specific activity against glycylprolyl-4-nitroaniline of 37.5 U/mg and an enzyme concentration of 1.41 mg/ml in the stock solution was used.
100 μl test compound in a concentration range of 1*10−5 M–1*10−8 M respectively were admixed with 50 μl glycylprolyl-4-nitroaniline in different concentrations (0.4 mM, 0.2 mM, 0.1 mM, 0.05 mM) and 100 μl HEPES (40 mM, pH7.6; ion strength=0.125). The assay mixture was pre-incubated at 30° C. for 30 min. After pre-incubation, 20 μl DPIV (1:600 diluted) was added and measurement of yellow color development due to 4-nitroaniline release was performed at 30° C. and λ=405 nm for 10 min. using a plate reader (HTS7000 plus, Applied Biosystems, Weiterstadt, Germany).
The Ki-values were calculated using Graphit version 4.0.13, 4.0.13 and 4.0.15 (Erithacus Software, Ltd, UK).
7.1 Results—Ki Values of DPIV Inhibition
100 μl inhibitor stock solution were mixed with 100 μl buffer (HEPES pH7.6) and 50 μl substrate (Gly-Pro-pNA, final concentration 0.4 mM) and preincubated at 30° C. Reaction was started by addition of 20 μl purified porcine DPIV. Formation of the product pNA was measured at 405 nm over 10 min using the HTS 7000Plus plate reader (Perkin Elmer) and slopes were calculated. The final inhibitor concentrations ranged between 1 mM and 30 nM. For calculation of IC50 GraFit 4.0.13 (Erithacus Software) was used.
8.1 Results—Determination of IC50 Values
DP II (188.8.131.52) releases N-terminal dipeptides from oligopeptides if the N-terminus is not protonated (McDonald, J. K., Ellis, S. & Reilly, T. J., 1966, J. Biol. Chem., 241, 1494–1501). Pro and Ala in P1-position are preferred residues. The enzyme activity is described as DPIV-like activity, but DP II has an acidic pH-optimum. The enzyme used was purified from porcine kidney.
100 μl glutaminyl pyrrolidine or glutaminyl thiazolidine in an concentration range of 1*10−4 M–5*10−8 M were admixed with 100 μl μl buffer solution (40 mM HEPES, pH7.6, 0.015% Brij, 1 mM DTT), 50 μl lysylalanylaminomethylcoumarine solution (5 mM) and 20 μl porcine DP II (250fold diluted in buffer solution). Fluorescence measurement was performed at 30° C. and λexiatation=380 nm, λemission=465 nm for 25 min using a plate reader (HTS7000plus, Applied Biosystems, Weiterstadt, Germany). The Ki-values were calculated using Graphit 4.0.15 (Erithacus Software, Ltd., UK) and were determined as Ki=8.52*10−5 M±6.33*10−6 M for glutaminyl pyrrolidine and Ki=1.07*10−5 M±3.81*10−7 M for glutaminyl thiazolidine.
Glutaminyl pyrrolidine and glutaminyl thiazolidine were tested for their cross reacting potency against dipeptidyl peptidase I, prolyl oligopeptidase and prolidase.
Dipeptidyl Peptidase I (DP I, Cathepsin C):
DP I or cathepsin C is a lysosomal cysteine protease which cleaves off dipeptides from the N-terminus of their substrates (Gutman, H. R. & Fruton, J. S., 1948, J. Biol: Chem., 174, 851–858). It is classified as a cysteine protease. The enzyme used was purchased from Qiagen (Qiagen GmbH, Hilden, Germany). In order to get a fully active enzyme, the enzyme was diluted 1000fold in MES buffer pH5.6 (40 mM MES, 4 mM DTT, 4 mM KCl, 2 mM EDTA, 0.015% Brij) and pre-incubated for 30 min at 30° C.
50 μl glutaminyl pyrrolidine or glutaminyl thiazolidine in a concentration range of 1*10−5 M–1*10−7 M were admixed with 110 μl buffer-enzyme-mixture. The assay mixture was pre-incubated at 30° C. for 15 min. After pre-incubation, 100 μl histidylseryl-β-nitroaniline (2*10−5M) was added and measurement of yellow color development due to β-nitroaniline release was performed at 30° C. and λexcitation=380 nm, λemission=465 nm for 10 min., using a plate reader (HTS7000 plus, Applied Biosystems, Weiterstadt, Germany).
The IC50-values were calculated using Graphit 4.0.15 (Erithacus Software, Ltd., UK). No inhibition of the DP I enzyme activity by glutaminyl pyrrolidine or glutaminyl thiazolidine was found.
Prolyl Oligopeptidase (POP)
Prolyl oligopeptidase (EC 184.108.40.206) is a serine type endoprotease which cleaves off peptides at the N-terminal part of the Xaa-Pro bond (Walter, R., Shlank, H., Glass, J. D., Schwartz, I. L. & Kerenyi, T. D., 1971, Science, 173, 827–829). Substrates are peptides with a molecular weight up to 3000 Da. The enzyme used was a recombinant human prolyl oligopeptidase. Recombinant expression was performed in E. coli under standard conditions as described elsewhere in the state of the art.
100 μl glutaminyl pyrrolidine or glutaminyl thiazolidine in an concentration range of 1*10−4 M–5*10−8 M were admixed with 100 μl μl buffer solution (40 mM HEPES, pH7.6, 0.015% Brij, 1 mM DTT) and 20 μl POP solution. The assay mixture was pre-incubated at 30° C. for 15 min. After pre-incubation, 50 μl glycylprolylprolyl-4-nitroaniline solution (0.29 mM) was added and measurement of yellow color development due to 4-nitroaniline release was performed at 30° C. and λ=405 nm for 10 min using a plate reader (sunrise, Tecan, Crailsheim, Germany). The IC50-values were calculated using Graphit 4.0.15 (Erithacus Software, Ltd., UK). No inhibition of POP activity by glutaminyl pyrrolidine or glutaminyl thiazolidine was found.
Prolidase (X-Pro Dipeptidase)
Prolidase (EC 220.127.116.11) was first described by Bergmann & Fruton (Bergmann, M. & Fruton, J S, 1937, J. Biol. Chem. 189–202). Prolidase releases the N-terminal amino acid from Xaa-Pro dipeptides and has a pH optimum between 6 and 9.
Prolidase from porcine kidney (ICN Biomedicals, Eschwege, Germany) was solved (1 mg/ml) in assay buffer (20 mM NH4(CH3COO)2, 3 mM MnCl2, pH 7.6). In order to get a fully active enzyme the solution was incubated for 60 min at room temperature.
450 μl glutaminyl pyrrolidine or glutaminyl thiazolidine in an concentration range of 5*10−3 M–5*10−7 M were admixed with 500 μl buffer solution (20 mM NH4(CH3COO)2, pH 7.6) and 250 μl IIe-Pro-OH (0.5 mM in the assay mixture). The assay mixture was pre-incubated at 30° C. for 5 min. After pre-incubation, 75 μl Prolidase (1:10 diluted in assay buffer) were added and measurement was performed at 30° C. and λ=220 nm for 20 min using a UV/Vis photometer, UV1 (Thermo Spectronic, Cambridge, UK).
The IC 50-values were calculated using Graphit 4.0.15 (Erithacus Software, Ltd., UK). They were determined as IC50>3 mM for glutaminyl thiazolidine and as IC50=3.4*104M±5.63*10−5 for glutaminyl pyrrolidine.
Male Wistar rats (Shoe: Wist(Sho)) with a body weight ranging between 250 and 350 g were purchased from Tierzucht Schönwalde (Schönwalde, Germany).
Animals were single-caged under conventional conditions with controlled temperature (22±2° C.) on a 12/12 hours light/dark cycle (light on at 06:00 AM). Standard pelleted chow (ssniff® Soest, Germany) and tap water acidified with HCl were allowed ad libitum.
Catheter Insertion into Carotid Artery
After ≧one week of adaptation at the housing conditions, catheters were implanted into the carotid artery of Wistar rats under general anaesthesia (i.p. injection of 0.25 ml/kg b.w. Rompun® [2%], BayerVital, Germany and 0.5 ml/kg b.w. Ketamin 10, Atarost GmbH & Co., Twistringen, Germany). The animals were allowed to recover for one week. The catheters were flushed with heparin-saline (100 IU/ml) three times per week. In case of catheter dysfunction, a second catheter was inserted into the contra-lateral carotid artery of the respective rat. After one week of recovery from surgery, this animal was reintegrated into the study. In case of dysfunction of the second catheter, the animal was withdrawn from the study. A new animal was recruited and the experiments were continued in the planned sequence, beginning at least 7 days after catheter implantation.
Rats with intact catheter function were administered placebo (1 ml saline, 0.154 mol/l) or test compound via the oral and the intra-vasal (intra-arterial) route. After overnight fasting, 100 μl samples of heparinised arterial blood were collected at −30, −5, and 0 min. The test substance was dissolved freshly in 1.0 ml saline (0.154 mol/l) and was administered at 0 min either orally via a feeding tube (75 mm; Fine Science Tools, Heidelberg, Germany) or via the intra-vasal route. In the case of oral administration, an additional volume of 1 ml saline was injected into the arterial catheter. In the case of intra-arterial administration, the catheter was immediately flushed with 30 μl saline and an additional 1 ml of saline was given orally via the feeding tube.
After application of placebo or the test substances, arterial blood samples were taken at 2.5, 5, 7.5, 10, 15, 20, 40, 60 and 120 min from the carotid catheter of the conscious unrestrained rats. All blood samples were collected into ice cooled Eppendorf tubes (Eppendorf-Netheler-Hinz, Hamburg, Germany) filled with 10 μl 1M sodium citrate buffer (pH 3.0) for plasma DPIV activity measurement. Eppendorf tubes were centrifuged immediately (12000 rpm for 2 min, Hettich Zentrifuge EBA 12, Tuttlingen; Germany): The plasma fractions were stored on ice until analysis or were frozen at −20° C. until analysis. All plasma samples were labelled with the following data:
The assay mixture for determination of plasma DPIV activity consisted of 80 μl reagent and 20 μl plasma sample. Kinetic measurement of the formation of the yellow product 4-nitroaniline from the substrate glycylprolyl-4-nitroaniline was performed at 390 nm for 1 min at 30° C. after 2 min pre-incubation at the same temperature. The DPIV activity was expressed in mU/ml.
Statistical evaluations and graphics were performed with PRISM® 3.02 (GraphPad Software, Inc.). All parameters were analysed in a descriptive manner including mean and SD.
11.1 Results—In Vivo DPIV-Inhibition at tmax
Side chain-modified glutamyl thiazolidines having a structure H-Glu(X)-Thia were synthesised, with polyethylene glycol or glycine oligomers of various chain lengths being used as X (see Method A of example for description of synthesis). The binding characteristics of those derivatives and their transportability by the peptide transporter PepT1 were investigated.
Surprisingly, it was found that the side chain modifications alter the binding characteristics of the compounds to DPIV only to a slight extent. In contrast, the ability of the inhibitors to be transported by the peptide transporter is dramatically diminished by the side chain modification.
Side chain modified inhibitors of DPIV or DPIV-like enzymes are therefore well suited to achieving site directed inhibition of DPIV in the body.
12.1 Results: Transportability of Selected DPIV-Inhibitors.
Using a novel in vivo adhesion assay which takes advantage of vital dye labeled tumor cells and their detection in the target tissue in situ (von Hörsten et al, 2000; Shingu et al., 2002), the current example investigates whether the in vivo adhesion of MADB106 tumor cells differs in DPIV in treated wild type F344 rats and F344 substrains with a mutation of the DPIV gene.
Animals, Injection of Tumor Cells and Processing of Lungs
F344USA, F344JAP and F344GER substrains were obtained from a breeding colony at the Central Animal Laboratory at Hannover Medical School, Germany. All substrains were bred for one generation and maintained in a specific-pathogen-free facility at 25° C. under a 12 h light–12 h dark cycle (light on at 07.00 h), with ad libitum access to food and water. The exact number of animals used per experiment is indicated by the F values with at least four animals per condition and time point.
Cell culture, injection of tumor cells, dissection of the animals and immunohistochemistry were conducted as previously described (von Hörsten et al., 2000). In brief, 1×106 MADB106 tumor cells derived from log phase of tumor growth were injected via the lateral tail vein and lungs removed at different time points thereafter. For in situ quantification of tumor cells at early time points after injection (30 min), cells were vital dye stained using the fluorescein derivate 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) before injection. For quantification of lung surface colonies at later time points (2 weeks after tumor cell inoculation), en-bloc dissected lungs and the heart were injected with 8 ml Bouin's solution (72% saturated picric acid solution, 23% formaldehyde, and 5% glacial acetic acid) and fixed in the same solution until lung surface nodules were counted (see below).
Four experiments were conducted:
Effect of a single injection of isoleucyl thiazolidine fumarate (2 mg i.v.+isoleucyl thiazolidine fumarate 2 mg i.p.) on lung tumor colonization in F344USA wild-type rats;
Effect of single injection of isoleucyl thiazolidine fumarate 2 mg i.v.+isoleucyl thiazolidine fumarate 2 mg i.p. on tumor cell adhesion to lungs of F344JAP, F344GER and F344USA rats;
Effect of single injection of isoleucyl cyanopyrrolidine (0.1 mg i.v.+0.1 mg i.p.) on tumor cell adhesion to lungs of F344USA wild-type rats;
Effect of single injection of valyl-pyrrolidine fumarate (0.1 mg i.v.+0.1 mg i.p.) on tumor cell adhesion to lungs of F344USA wild-type rats;
Immunohistochemistry of CFSE-Labeled Tumor Cells in Lungs
Immunostaining of CFSE-labeled MADB106 tumor cells was achieved using mAb characterizing the intracellular CFSE antigen (anti-CFSE; mAb DE1, Boehringer, Mannheim, Germany; mouse, 1:100). For immunohistochemistry, one or two consecutive APAAP stainings were performed as previously described (von Hörsten et al, 2000; Shingu et al., 2002). Control sections were included in which one or both primary antibodies were omitted.
Quantification of Tumor Targets: In Vivo/In Situ Cell Adhesion Assay
Vital dye (Carboxyfluorescein; CFSE) labeling of MADB106 tumor cells allows the quantification of tumor cells and NK cells in thick sections of lung tissue by stereology in situ (von Hörsten et al, 2000). In the present study we produced thin sections (8 μm) of the same lungs (n=10) and performed additional microscopic counting by image analysis of DE1 positive cells. This was done to further simplify the previously validated stereological quantification technique. Therefore, in the present study, the assessment of DE1 positive tumor cells in lung tissue from different substrains 30 min after tumor inoculation was carried out using image analysis approach. All CFSE-labeled MADB106 tumor cells and leukocyte subsets within a grid on the ocular lens were counted (Zeiss Kpl-W 12.5×; grid 0.75×0.75 mm=0.5625 mm2/grid, using a Zeiss Neofluar objective, ×10, NA=0.3). Each right upper lobe of the lungs was sectioned at 6 randomly chosen non-adjacent levels. From each level, three sections were evaluated. On average, 30 grid numbers per section were examined (i.e. 0.5625 mm2/grid×30 grids×3 sections×6 levels) resulting in an area per animal of 3.04 cm2.
Quantification of Macrometastasis on Lungs
For quantification of lung surface colonies at later time points (2 weeks after tumor cell inoculation), en-bloc dissected lungs and the heart were injected with 8 ml Bouin's solution (72% saturated picric acid solution, 23% formaldehyde, and 5% glacial acetic acid) and fixed in the same solution until lung surface nodules were counted. Three areas per lungs were examined using a gauge (1 cm2) and lung surface colony numbers were expressed as mean/cm2 according to the method of Wexler (Wexler, 1966).
Data from in vivo adhesion assay were analyzed by one-way ANOVAs and Fisher's PLSD post hoc tests, if appropriate. An asterisk indicates significant post hoc effects vs. saline (SHAM) treated controls obtained by Fisher's PLSD. All data are presented as means±S.E.M.
Effect of a Single Injection of Isoleucyl Thiazolidine Fumarate on Lung Tumor Colonization in F344USA Rats
The number of lung surface tumor colonies after single isoleucyl thiazolidine fumarate administration in F344 rats 2 weeks after injection of MADB106 tumor cells is illustrated in
Effect of Single Injection of Isoleucyl Thiazolidine Fumarate on Tumor Adhesion in F344JAP, F344GER and F344USA Rats
The mean number of CFSE positive cells in lung tissue at 30 min after inoculation of MADB106 tumor cells in the three substrains is illustrated in
Effect of Single Injection of Isoleucyl Cyanopyrrolidine TFA on Tumor Adhesion of F344USA Rats
The mean number of CFSE positive cells in lung tissue at 30 min after inoculation of MADB106 tumor cells after isoleucyl cyanopyrrolidine TFA treatment is illustrated in
Effect of Single Injection of Valyl Pyrrolidine Fumarate on Tumor Adhesion of F344USA Rats
The mean number of CFSE positive cells in lung tissue at 30 min after inoculation of MADB106 tumor cells after valyl pyrrolidine fumarate treatment is illustrated in
Tumor cell adhesion and colonization is significantly modified by single injection of isoleucyl thiazolidine fumarate only in mutant F344 substrains suggesting an interaction of the ligand with mutant DPIV and tumor cells. Since the ligand did not significantly affect tumor adhesion in wild type F344USA rats, this may indicate that compound is not interacting with the binding site of MADB106 tumor cells.
In the previous example it was demonstrated that MADB106 tumor cell adhesion is significantly modified by a single administration of isoleucyl thiazolidine fumarate only in mutant F344 substrains but not in wild type DPIV expressing F344USA rats. DPIV inhibitors/ligands may interact with the growth of tumor metastases exhibiting properties similar to chemotherapeutic compounds and/or immunotherapeutical compounds. In contrast to that, the current example investigates whether the tumor colonization of MADB106 tumor cells differs in chronically DPIV-inhibitor treated wild type F344 rats.
Animals, Injection of Tumor Cells and Processing of Lungs
F344USA rats were obtained from a breeding colony at the Central Animal Laboratory at Hannover Medical School, Germany. All rats were bred at least for one generation and maintained in a specific-pathogen-free facility at 25° C. under a 12 h light–12h dark cycle (light on at 07.00 h), with ad libitum access to food and water. The exact number of animals used per experiment is indicated by the F values with at least four animals per condition and time point.
Cell culture, injection of tumor cells, dissection of the animals and immunohistochemistry were conducted as previously described (von Hörsten et al., 2000; Shingu et al., 2002). In brief, 1×106 MADB106 tumor cells derived from log phase of tumor growth were injected via the lateral tail vein and lungs removed at different time points thereafter. For in situ quantification of tumor cells at early time points after injection (30 min), cells were vital dye stained using the fluorescein derivate 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) before injection. For quantification of lung surface colonies at later time points (2 weeks after tumor cell inoculation), en-bloc dissected lungs and the heart were injected with 8 ml Bouin's solution (72% saturated picric acid solution, 23% formaldehyde, and 5% glacial acetic acid) and fixed in the same solution until lung surface nodules were counted (see below).
Two experiments were conducted:
Effect of chronic infusion of different dosages of isoleucyl thiazolidine fumarate (0 mg, 0.04 mg, 0.4 mg, 4 mg/24 h intragastral via implanted osmotic minipumps) on body weight change and lung tumor colonization in F344USA wild-type rats
Effect of chronic infusion of isoleucyl thiazolidine fumarate (4 mg/24 h intragastral via implanted osmotic minipumps), lisoleucyl cyanopyrrolidine TFA (0.1 mg/24 h intragastral via implanted osmotic minipumps) and valyl pyrrolidine fumarate (0.1 mg/24 h intragastral via implanted osmotic minipumps) on lung tumor colonization in F344USA wild-type rats.
Implantation of Osmotic Minipumps for Chronic Intragastric Infusion of Compounds
Osmotic minipumps (Alzet model 2ML4; flow rate, 2.5 μl/hr; Alza Corporation), administering a constant supply of the different compounds, aseptically prefilled with either saline or DPIV inhibitor were placed subcutaneously in the abdominal area. Minipums were attached to a cannula via polyethylene tubing. The cannula was implanted intragastrically with a heating-induced enlarged tip of the cannula in the lumen of the gaster.
Quantification of Macrometastasis on Lungs
For quantification of lung surface colonies at later time points (2 weeks after tumor cell inoculation), en-bloc dissected lungs and the heart were injected with 8 ml Bouin's solution (72% saturated picric acid solution, 23% formaldehyde, and 5% glacial acetic acid) and fixed in the same solution until lung surface nodules were counted. Three areas per lungs were examined using a gauge (1 cm2) and lung surface colony numbers were expressed as mean/cm2 according to the method of Wexler (Wexler, 1966).
Data from in vivo body weight gain and number of lung surface tumor colonies were analyzed by one-way ANOVAs and Fisher's PLSD post hoc tests, if appropriate. An asterisk indicates significant post hoc effects vs. saline (SHAM) treated controls obtained by Fisher's PLSD. All data are presented as means±S.E.M.
Effect of Chronic Infusion of Isoleucyl Thiazolidine Fumarate (0 mg, 0.04 mg, 0.4 mg, 4 mg/24 h) on Lung Tumor Colonization
The change of body weight after chronic infusion of different dosages of isoleucyl thiazolidine fumarate in F344 rats 2 weeks after injection of MADB106 tumor cells is illustrated in
The number of lung surface tumor colonies after chronic infusion of different dosages of isoleucyl thiazolidine fumarate in F344 rats 2 weeks after injection of MADB106 tumor cells is illustrated in
Effect of Chronic Infusion of Isoleucyl Thiazolidine Fumarate, Isoleucyl Cyanopyrrolidine TFA, and Valyl Pyrrolidine Fumarate on Lung Tumor Colonization
The number of lung surface tumor colonies after chronic infusion of isoleucyl thiazolidine fumarate; isoleucyl cyanopyrrolidine TFA, and valyl pyrrolidine fumarate in F344 rats 2 weeks after injection of MADB106 tumor cells is illustrated in
Metastasis of MADB106 is reduced by chronic treatment using different DPIV Inhibitors (isoleucyl thiazolidine fumarate; isoleucyl cyanopyrrolidine TFA) suggesting protective-like class effects. Possibly, isoleucyl thiazolidine fumarate and isoleucyl cyanopyrrolidine TFA protect from metastasis either via interaction with cell adhesion processes or via a modification of the cellular host defense mechanisms. It is also possible that DPIV inhibitor treatment exhibits cytostatic effects. These antimetastic effects substantiate the biological properties of DPIV Inhibitors for the treatment of cancer and metastatic disease.